Disease and epidemiology: Chikungunya is a physically debilitating disease of humans mainly in Africa and Asia. The symptoms include abrupt onset of high fever, rash or hemorrhages, arthralgia and occasional involvement of the nervous system, heart and liver. The incapacitation is due to arthralgia, which can persist for years (Sarkar et al., 1965; Rao et al., 1965; Nimmannitya et al., 1969; Schuffenecker et al., 2006). The disease is caused by Chikungunya virus (CHIKV), and is spread by Aedes spp. mosquitoes, either through other forest-dwelling vertebrate hosts (in Africa) or by a human-mosquito-human cycle (in Asia) (Powers et al., 2000). There have been several major outbreaks of the disease, including the recent ones in the Indian Ocean, Malaysia and India, with several thousands of people afflicted. In India, the major outbreaks appear to have occurred once in the 1960s and then in 2005-2006 (Shah et al., 1964; Rao et al., 1965; Chaturvedi et al., 1970; Ravi, 2006). In the recent outbreak, several districts in Karnataka, Andhra Pradesh, Tamil Nadu, Maharashtra, and possibly Orissa have been affected. The disease can be diagnosed by various serological tests, but definitive identification requires verification of the genetic material since many closely related arboviruses cause similar disease. Treatment is only palliative and there is no commercially available vaccine.
The virus harbors a single-stranded, positive sense RNA genome, and belongs to group A arboviruses along with Sindbis and Semliki Forest disease viruses in the alphavirus genus of Togaviridae family (Fauquet et al., 2005). The virion is 50-60 nm in size and is inactivated by 70% ethanol, 1% sodium hypochlorite, 2% glutaraldehyde, lipid solvents, moist or dry heat >58° C., as well as drying.
Genotyping suggests the existence of three clades: West African, East-Central-South-African and Asian. The Asian and African strains form closely-related clades that differ from each other in sequence, antigenicity and virus properties. The Asian isolates appear to be more conserved than either of the African clades (Powers et al., 2000; Schuffenecker et al., 2006). The recent Indian Ocean outbreak isolates show a characteristic change from Alanine to Valine in position 226 of the envelope glycopriten E1 from early to later phase of the disease, respectively (Schuffenecker et al., 2006). While the importance of this is not understood, an evolutionary advantage for the virus can be surmised.
Not much is known about the viral proteins, their function or pathogenicity. The genome consists of ˜12 kilobases, with a 5′ 7 mG cap and the 3′ poly(A) region, and a base composition of 30% A, 25% C and G, and 20% U. The genome has the sequence of 5′-nsP1-nsP2-nsP3-nsP4-(junction region)-C-E3-E2-6K-E1-polyA-3′. The non-structural proteins are translated directly from the 5′ two-thirds of the genome, and the structural proteins are produced from the 26S subgenomic RNA which is collinear with the 3′ one-third of the genome. The genome contains conserved repeat sequences as well as an internal poly(A) tract within the 3′ non-translated region (Khan et al., 2002; Schuffenecker et al., 2006).
Based on sequence information, it has been deduced that nsP1, nsP2, nsP3, nsP4, C, E3, E2, 6K and E1 proteins contain 535, 798, 530, 611, 261, 64, 423 and 61 amino acids (Khan et al., 2002; Schuffenecker et al., 2006). The envelope proteins can be observed on SDS gels as 62 kD E2/E3, which is cleaved into E2 and E3 within 90 mins, and 45-50 kD E1 and E2 that migrate closely together. E1 and E2 associate tightly with each other rapidly. The 11 kD E3 protein is not associated with the virion, and is released into the medium (Simizu et al., 1984; Ranadive and Banerjee, 1990). The viral E1 glycoprotein agglutinates erythrocytes, and hemagglutination (HA) and hemagglutination inhibition (HI) tests can be performed routinely on goose erythrocytes for diagnostic purposes. HA activity of the virus is not susceptible to trypsin, and is enhanced by tween-ether treatment (Hannoun, 1968). Serum with HI titers of >40 generally shows neutralization capacity (Bedekar and Pavri, 1969b). Isolation of virus can be performed in newborn rats or mice, or in animal or insect cell cultures. Vero, African green monkey kidney, BHK21, BSC-1, chick embryo fibroblasts and C6/36 cells have been used for in vitro virus isolation and expansion. The virus replicates fairly rapidly in cell culture. Depending on dose and on the cell line, cytopathic effect can be observed in 12-48 hrs. At multiplicities of 1-5, following a 5-6 hr eclipse period, the intracellular virus titer rises sharply and reaches peak by 12 hrs. Extracellular virus can be observed at 8 hrs post-infection and peaks at 12-24 hrs depending on the cell system and dose of the inoculum (Chain et al., 1966; Higashi et al., 1967; Hahon and Hankins, 1970; Eckels et al., 1970). Spread of infection through the monolayer differs in different cell types, involving both extracellular and cell-to-cell transmission in BHK21 cells, but only the former in L929 and guinea pig lung cells (Hahon and Zimmerman, 1970). Titers in supernatants can reach as high as that observed with mouse brain preparations (Shah et al., 1964; Paul and Singh, 1968; Umrigar and Kadam, 1974).
The virus can be concentrated from cell culture supernatant by ultracentrifugation, or precipitation with ammonium sulphate, alum, or polyethylene glycol (Eckels et al., 1970; Klein et al., 1970; Banerjee and Ranadive, 1988; Killington et al., 1996). The virus can be further purified by using rate zonal centrifugation, equilibrium density gradient or gel filtration (Eckels et al., 1970; Simizu et al., 1984; Banerjee and Ranadive, 1988). Titration of the virus can be performed by immunofluorescence, ELISA, complement fixation, agar gel immunodiffusion, hemagglutination and inhibition, plaque assays, or neutralization.
It is unknown how CHIKV or other alphaviruses cause arthritis. Suggested mechanisms include replication leading to cell death and tissue damage, immune-mediated attack on the joints, or immune-complex-mediated inflammation. While Semliki forest virus and Ross River virus have been shown to known to replicate in bone-associated connective tissue in neonates as well as skin and muscle in adult mice (Heise et al., 2000), no such study has been done with CHIKV.
In contrast to Dengue virus, which requires adaptation for infection of animals, CHIKV shows rapid and high fatality on primary inoculation of clinical samples (Myers et al., 1965). However, newborn mice and rats are the only species that show disease (Chakravarthy and Sarkar, 1969). In a dose-dependent manner, rat/mouse pups show illness and high mortality following intracerebral, intraperitoneal or subcutaneous inoculation of patient sera in 3-10 days, yielding 105.5-107.0 mouse-LD50 of virus per mL of serum (Shah et al., 1964). Animals rapidly manifest sluggishness, severe inappetence, cyanosis, dermal hypothermia, and death. Pathologically, they show cardiac enlargement, hemorrhages in gastrointestinal tract, alveoli, bladder, joints and skin, skeletal muscle and fat pad necrosis, and diffuse intestinal dysfunction (Nimmannitya et al., 1969; Weiss et al., 1965). Survivors (injected lower doses) show stunted growth, but develop HI antibodies and are protected from challenge after intracerebral or intraperitoneal challenge (Shah et al., 1964; Giovarelli et al., 1977). Newborn bunnies and guinea pigs are moderately susceptible with some virus recovery, and one-day kitten are less susceptible. Adult mice, rats, guinea pigs, hamsters, hare and rabbits show viremia, but not disease, and develop HI and neutralizing antibodies, whereas adult cats and fowl don't. Low titers of HI antibodies without viremia or neutralizing antibodies can be seen in cows, sheep, goats and horses (McIntosh et al., 1963; Bedekar and Pavri, 1969a; Chakravarthy and Sarkar, 1969). The susceptibility of birds is controversial. In one study, white leghorn chicks have been shown to succumb to virus inoculation, and recovered birds develop neutralization antibodies (Bedekar and Pavri, 1969a). This and other studies involving chicken, sparrows, pigeons and bats have shown seroconversion without viremia or nothing (Shah et al., 1964; Bedekar and Pavri, 1969a).
Adult monkeys belonging to various species show viremia, can transmit virus to mosquitoes, and develop long lasting neutralizing antibodies (Paul and Singh, 1968). Wild monkeys and baboons in Africa circulate high titres of the virus without any apparent sickness as a result of infection, and can transmit the virus to through mosquitoes (McIntosh et al., 1963). However, serological evidence of natural infection of monkeys does not exist in India (Bedekar and Pavri, 1969b).
CHIKV infection (whether clinical or silent) is thought to confer life-long immunity. Because of close antigenic relationship, cross-protection between different strains (Casals, 1957; Porterfield, 1961; Shah et al., 1964) as well as reciprocal cross-protection among other alphaviruses (Parks and Price, 1958; Hearn and Rainey, 1963) can be hypothesized, and is demonstrated in animal models. However, there is some evidence that live attenuated alphavirus vaccines may interfere with a subsequent, related vaccine (McClain et al., 1998).
As a prelude to vaccines, initial CHIKV preparations involved either formalin inactivation (Harrison et al., 1967) or tween-ether extraction of virus grown in vitro (Eckels et al., 1970). While formalin kills HA activity, the latter treatment retains the HA activity completely, although both lose infectivity drastically. However, they both elicit similar HA and complement-fixing and neutralization antibodies and also show similar levels of protection in lethal challenge studies (Eckels et al., 1970).
US Army Medical Institute of Infectious Diseases in Fort Detrick, Md. made a candidate vaccine for CHIKV. CHIKV strain 15561 from Thailand (1962 outbreak) was used to develop a small lot of green monkey passaged, formalin-inactivated preparation that was administered to 16 volunteers who showed high immune responses and no adverse effects (Harrison et al., 1971). The GMK-passaged virus was further serially passaged by plaquing 18 times in MRC-5 cells (Levitt et al., 1986), and found to be safe and immunogenic in phase I trial with 15 alphavirus-naïve individuals, viremia occurring on day 2-4 post-inoculation (McClain et al., 1988). In a randomized, double-blind, placebo-controlled, phase II trial, 73 alphavirus-naïve volunteers of 18-40 years were injected with 0.5 mL dose containing either ˜105 PFU of virus (59 subjects) or placebo (14 subjects) subcutaneously. Serological evaluation involved plaque reduction neutralization titer (PRNT), and a 50% reduction titer of ≧20 was considered positive. Local and systemic reactions were limited to vaccine take whereas 8% of CHIKV vaccinees (and none of placebo group) showed arthralgia. 98.3% of vaccinates seroconverted by day 28, achieving peak PRNT50 titers of 1:10240 at 28-42 days. Although antibody levels declined somewhat over time, 85% of the vaccinees were still seropositive at one year, with titers of 1:1280 at 180-360 days (Edelman et al., 2000).